GSM6685505: Rat Sciatic Nerve, dissociated, CCI Day 12, Replicate 7b; Rattus norvegicus; RNA-Seq
Enzymatic digestion: About 10 mm of naïve sciatic nerve or 10 mm of the CCI ligated sciatic nerve was dissected from rats transcardially perfused with PBS for immediate enzymatic dissociation. Nerve tissues were finely chopped with scalpels and then digested in 1-2 ml enzyme cocktail comprising: trypsin (Gibco, 3 mg/ml), collagenase II (Worthington 800U/ml), hyaluronidase (Worthington, 1105 U/ml), pronase (Sigma, 240 U/ml) and dispase (Sigma, 5 U/ml) in HEPES buffered HBSS with 0.45% glucose. The digestion continued at 37°C and 5% CO2, and was terminated after 40-60 min by adding 1-2 ml dissociation medium (HBSS with HEPES, 0.45% glucose and 1% BSA) followed by trituration with a p1000 pipette. The dissociate was then filtered over a 40 um filter and into a 50 ml tube on ice and 10 ml dissociation medium was added. Cells were centrifugated (10 min, 1000rpm, 10°C) and resuspended in 1 ml dissociation medium for an additional two washes. Cells were finally resuspended in PBS with 1% BSA and counted. Single cell RNA-sequencing was performed using the Chromium Single Cell 3′ Kit with v2 chemistry (10X Genomics) according to manufacturer instructions. We targeted 8000 cells per 10X GEM reaction. Libraries were QC'ed by verifying optimal size using the High Sensitivity DNA Agilent Bioanalyzer kit and cDNA concentration quantified using the Invitrogen Qubit HS DNA kit according to the manufacturer's instructions. Then libraries were sequenced on an Illumina Novaseq 6000.